Replicative Acinetobacter baumannii strains interfere with phagosomal maturation by modulating the vacuolar pH

Bacterial pneumonia is a common infection of the lower respiratory tract that can afflict patients of all ages. Multidrug-resistant strains of Acinetobacter baumannii are increasingly responsible for causing nosocomial pneumonias, thus posing an urgent threat. Alveolar macrophages play a critical role in overcoming respiratory infections caused by this pathogen. Recently, we and others have shown that new clinical isolates of A. baumannii, but not the common lab strain ATCC 19606 (19606), can persist and replicate in macrophages within spacious vacuoles that we called Acinetobacter Containing Vacuoles (ACV). In this work, we demonstrate that the modern A. baumannii clinical isolate 398, but not the lab strain 19606, can infect alveolar macrophages and produce ACVs in vivo in a murine pneumonia model. Both strains initially interact with the macrophage endocytic pathway, as indicated by EEA1 and LAMP1 markers; however, the fate of these strains diverges at a later stage. While 19606 is eliminated in an autophagy pathway, 398 replicates in ACVs and are not degraded. We show that 398 reverts the natural acidification of the phagosome by secreting large amounts of ammonia, a by-product of amino acid catabolism. We propose that this ability to survive within macrophages may be critical for the persistence of clinical A. baumannii isolates in the lung during a respiratory infection.


Introduction
The opportunistic bacterial pathogen Acinetobacter baumannii is an urgent global public health threat. This pathogen is associated with a wide variety of nosocomial infections including pneumonia, meningitis, urinary tract infection, and wound infection. However, respiratory tract infections are the principal disease associated with A. baumannii [1]. Alveolar macrophages (AMs) and lung epithelial cells are the first line of defense against respiratory pathogens [2,3]. Macrophages are particularly important during the early stages of A. baumannii respiratory infection, as they initiate and regulate the innate immune response against A. baumannii by recruiting non-resident macrophages and neutrophils [4][5][6]. Consequently, depletion of AMs during A. baumannii infection results in an increased in bacterial burden, tissue damage, and sepsis in the host [7]. Moreover, it has been demonstrated that murine AMs can internalize A. baumannii in vivo at 4 h post intranasal infection [5].
Phagocytosis is a mechanism by which cells internalize large particles, such as apoptotic cells or bacteria. Phagocytosis is required to maintain cellular homeostasis and is essential in the innate immune response against pathogens [8,9]. This process is performed primarily by myeloid cells including macrophages, neutrophils, monocytes, and dendritic cells [10,11]. After particle recognition and internalization, the newly formed phagosome undergoes a dynamic series of steps called "maturation". Each phase of the process is determined by the presence of specific signaling molecules located on the phagosomal membrane. Initially, the phagosome recruits the characteristic endosomal marker Rab5 with its effectors EEA1 and VPS34. Maturation continues with the exchange of these markers by late endosome-associated molecules, such as Rab7, HOPS complex, LAMP1 and LAMP2 among others [12][13][14]. The process concludes with the formation of the phagolysosome and the degradation of its internal content by the activity of proteases, nucleases and lipases [15][16][17]. During maturation phagosomes progressively decrease luminal pH, mainly through proton pump Vacuolar ATPases (V-ATPase) activity [18,19]. This acidification is critical to halt growth of pathogenic microorganisms and is necessary for diverse microbicidal functions of the phagosome that depend on H + concentrations, such as activation of degradative enzymes and production of reactive oxygen species (ROS) [20,21].
Many pathogenic bacteria have developed various strategies to hijack the host machinery, avoid phagosome maturation, and produce a specific compartment where intracellular bacteria can survive and replicate [22,23]. Brucella abortus, Legionella pneumophila and Chlamydia trachomatis escape early from the endocytic pathway and produce unique vacuoles that contain endoplasmic reticulum and golgi membrane markers [24][25][26]. Mycobacterium tuberculosis and Salmonella block phagosome maturation and arrest the bacterial-containing vacuole at an early stage [27][28][29]. Alternatively, Coxiella burnetii interacts with the endocytic pathway to develop a replication-permissive spacious vacuole that is similar to a phagolysosome [30,31]. A. baumannii, on the other hand, has classically been considered an extracellular pathogen. Recent reports, however, have demonstrated that A. baumannii clinical isolates, but not "domesticated lab strains", can replicate in vitro within Acinetobacter Containing Vacuoles (ACVs) in lung epithelial cells and macrophages [32,33]. The trafficking pathways involved in the biogenesis of these ACV remains unknown.
In this work, we demonstrate that the modern clinical isolate of A. baumannii, 398, can survive and replicate inside AM ACVs during a murine pulmonary infection. Moreover, we characterize the intracellular trafficking of A. baumannii and describe key differences between the "domesticated lab strain" 19606 and the new clinical isolate 398 in J774A.1 macrophages. We additionally demonstrate that the replicative strain (strains that can replicate in vitro in eukaryotic cells) generates a non-degradative ACV. Finally, we provide evidence that resistance to acidic pH and secretion of ammonia, a by-product of amino acid degradation, are key factors for the A. baumannii intracellular survival.

The A. baumannii recent clinical isolate 398 develops ACVs in alveolar macrophages in vivo
Macrophages play a critical role in host immune defense against A. baumannii respiratory infections [5]. It has been reported that macrophages can phagocytose and degrade common lab strains of A. baumannii such as 19606, but diverse new clinical isolates persist and/or replicate in mouse and human macrophages [33,34]. We have previously demonstrated that A. baumannii new clinical isolates replicate in macrophages inside an Acinetobacter Containing Vacuole (ACV) [33]. Yet, to the best of our knowledge, there is no evidence showing that A. baumannii replicates inside macrophages in vivo. To analyze that, we evaluated the ability of two A. baumannii strains, the "domesticated lab strain" 19606 and the recent clinical isolate 398, to produce ACVs in macrophages during an acute lower respiratory tract infection into C57BL/6 mice. Both A. baumannii strains expressing GFP were each inoculated intranasally in C57BL/6 mice (*1 × 10 8 CFU), and after 3 and 24 hpi, the number of total and intracellular colony forming units (CFUs) present in the bronchoalveolar lavage fluid (BALF) was determined by antibiotic protection assays. Additionally, the number of total and infected AMs were analyzed using flow cytometry. Finally, the presence of ACVs in macrophages was established by confocal microscopy (Fig 1A). CFU enumeration demonstrated similar numbers of total bacteria for both A. baumannii strains at 3 hpi, indicating that similar numbers of bacteria reached the lung (S1A Fig). However, the number of intracellular bacteria was greater for 398 than for 19606 (Fig 1B, left). At 24 hpi, both the total and intracellular CFUs for 398 were significantly higher compared to 19606 (Fig 1B right). Similar results were found in the rest of the lung (S1A Fig). In agreement with Qiu et al., (2012) [5], we detected an slight increase in the number of AMs between 3 hpi and 24 hpi by flow cytometry, however at 24 hpi neutrophils were the most prevalent immune cells (S1B Fig). The total number of AMs present in the BALF was similar between mice infected with both A. baumannii strains, but the number of AMs infected with 398 persisted over time, while the ones infected with 19606 significantly decreased at 24 hpi ( Fig 1C). Visualization of the cells present in the BALF by confocal and transmission electron microscopy (TEM) showed that only 398 was able to produce large ACVs in AMs, while the "domesticated lab strain" 19606 remained contained in single-bacterium phagosomes or was degraded by 24 hpi (Figs 1D and S1C). Moreover, large 398 ACVs (vacuoles containing more than~10 bacteria) were found in more than 40% of the infected AMs, whereas no big vacuoles containing 19606 were observed at 24 hpi (S2 Fig). Together, these experiments demonstrate that, while a lab strain is rapidly cleared, the clinical isolate 398 can infect, persist and possibly multiply intracellularly inside ACVs in murine AMs in vivo. . Cell nuclei were stained with DAPI (blue), A baumannii 19606 or 398 were detected by GFP fluorescence (green), and SiglecF was immunolabeled with specific antibodies (red). Arrows indicate AMs. Scale bars: 10 μm. Representative transmission electron microscopy images with infected AMs from BALF are shown at the right. The arrow indicates 398 ACV. Scale bars: 1 μm. Statistical analyses were performed using the Mann-Whitney test, **p < 0,0021, ***p<0,0006 ****p < 0,0001. Hpi: hours post-infection. Nascent phagosomes interact with different compartments of the endocytic pathway to ultimately fuse with lysosomes and degrade their internal content. This sequential process is called "maturation" and is characterized by the recruitment of specific molecular markers to the phagosomal membrane that guide their traffic inside the cell [14]. However, several bacterial pathogens have evolved to manipulate the host cell and survive intracellularly [22,23]. To characterize the biogenesis of the ACVs, we utilized the early or late endosomal markers EEA1 (Early Endosomal Antigen 1) and LAMP1 (Lysosomal-associated membrane protein 1), respectively. Approximately, 80% of the ACVs of A. baumannii clinical isolate 398 colocalized with EEA1 as early as 15 min post infection (mpi), however, the percentage of colocalization of this marker decreased over time. At 60 mpi less than 5% of the ACVs were EEA1 positive (Figs 2A, 2B and S4). When we analyzed the distribution of the late endosomal marker LAMP1, most of the ACVs were decorated with this marker as early as 60 mpi (Figs 2B, 2C and S5A). Similar results were obtained with 19606 for LAMP1 endosomal marker (S5B and S5C Fig).
Interestingly, no differences in cytotoxicity levels, as measured by the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) to the extracellular media, were detected in cells infected with 398 or 19606 (S6 Fig). This data demonstrates that in macrophages the phagosomes produced by both strains initially follow a canonical maturation.

The autophagic protein LC3 is absent from ACVs of the replicative A. baumannii 398 strain
Autophagy is a process by which eukaryotic cells remove unwanted material from the cell cytosol [35]. Xenophagy is a highly selective type of autophagy used by the host cells to detect and eliminate intracellular pathogens [36,37]. Bacterial phagosomes can recruit the canonical autophagy marker, LC3 (microtubule-associated protein 1 light chain 3), inducing the compartment maturation and content degradation. However, the relationship between autophagy and intracellular microorganisms is complex [38]. Some intracellular bacteria as C. burnetii and Serratia marcescens survive in an autophagic vacuole while others, such as M. tuberculosis and L. pneumophila, avoid the interaction with autophagosomes [39][40][41][42][43]. However, the relationship between the autophagic pathway and A. baumannii remains unclear and controversial. In epithelial cells, vacuoles of the strain AB5075 colocalize with LC3 before bacterial killing; however, ACVs of replicative strains C4 or ABC141 do not recruit the autophagic marker [32,44]. In our previous work we demonstrated that, in J774A.1 macrophages, ACVs of the clinical isolate UPAB1 are LC3 negative [33]. We hypothesized that these differences could depend on the A. baumannii strain used in these studies. To corroborate this, the subcellular distribution of LC3 was analyzed in J774A.1 cells infected with our replicative isolate GFP-398 or the non-replicative GFP-19606. At 4 hpi, most of 19606 phagosomes were labeled with the autophagic marker ( Fig 3A bottom row, Figs 3B and S7). On the contrary, 398 ACVs were LC3 negative up to 6 hpi (Figs 3A and 3C and S7). These data suggest that while non-replicative strains such as 19606 are eliminated by autophagy, replicative strains like 398 avoid the interaction with the autophagic pathway.

The ACV of A. baumannii strain 398 is a non-degradative compartment
The final step of phagosomal maturation is the fusion with lysosomes to form the phagolysosomal compartment in which the degradation of the internal content takes place [16]. To determine if the ACV is a degradative compartment, we used the fluorogenic probe DQ-BSA Green, a self-quenched substrate that fluoresces green when it is degraded by acidic proteases in the phagolysosome. The lysosomes of both uninfected and infected macrophages exhibited green fluorescence, indicating that lysosomal biogenesis and functions were not impaired

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Interference of phagosomal maturation by Acinetobacter baumannii  Fig 4D). These data indicate that the 398 ACVs are non-degradative, suggesting that 398 can manipulate the canonical maturation of the phagosome.

Luminal pH of the ACV increases during infection
The two main characteristics of phagolysosomes are an acidic pH and the presence of hydrolytic enzymes [8]. Phagosome acidification is mainly achieved by V-ATPases that pump H + into the luminal space of this compartment. To test the effect of vacuolar acidification on A. baumannii intracellular replication, we used bafilomycin A1, a specific V-ATPase inhibitor.

Interference of phagosomal maturation by Acinetobacter baumannii
While strain 19606 was degraded under normal conditions, in the presence of bafilomycin A1, 19606 was able to replicate ( Fig 5A). Moreover, large ACVs with many GFP-19606 bacteria were observed by confocal microscopy when incubated with bafilomycin A1 (Fig 5B). Furthermore, 398 intracellular replication was higher in the presence of bafilomycin A1 (S9 Fig). These data suggest that the luminal pH of the ACV is key factor in thwarting intracellular replication of A. baumannii.
The ability of 398 to survive and replicate within ACVs prompted us to analyze their pH. LysoSensor is a dye that is internalized in the cell, and its fluorescence intensity is dependent on the pH of the compartment in which it resides (increased brightness at low pH).  (Fig 6C). Furthermore, no 19606 vacuoles can be detected at 24 hpi (S10A Fig, bottom row) suggesting the degradation of the intracellular bacteria. These results demonstrate that although 398 vacuoles are initially acidic this strain, but not 19606, can actively increase the ACV's luminal pH.

Intracellular replication relies on tolerance to acidic pH and ammonia production
Our data suggests that the ACV pH is key in controlling A. baumannii replication and that the clinical isolate 398 actively increases the pH of the ACV after initially surviving acidic conditions. Previous work observed that Acinetobacter calcoaceticus clinical isolates are able to survive at acidic conditions (pH 4 to 6), but growth was significantly reduced compared to

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Interference of phagosomal maturation by Acinetobacter baumannii neutral pH [45]. Also, A. baumannii was found to be more tolerant to pH stress than other Acinetobacter species such as A. nosocomialis, A. pittii and A. calcoaceticus [46]. We hypothesized that replicative strains of A. baumannii are more tolerant to acid stress than non-replicative bacteria and as a result they can grow at faster rates under acidic conditions. To analyze the

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Interference of phagosomal maturation by Acinetobacter baumannii relationship between intracellular replication and growth fitness at acidic pH, we employed the replicative strain 398 and the non-replicative strains 19606 (Fig 7A). We tested the ability of these strains to grow in buffered LB broth at pH 5, 6, or 7, as previously described. Although the two strains grew similarly at neutral pH (S11 Fig), 19606 had a lower growth rate than 398 at pH 5 ( Fig 7B). In non-buffered LB (pH �5), tested strains alkalinized the culture media to pH�8 after growth for 18 h. However, 398 required significantly less time to neutralize the media than the non-replicative strain. For example, 398 reaches a pH of �7 at 6 h, while 19606 requires �10 h to reach the same pH (Fig 7C). A. baumannii is unable to grow using most sugars individually [47], instead, it heavily relies on amino acids. To feed the TCA cycle, the αamino groups of the amino acids are removed with the concomitant production of ammonia, which is secreted to the media, resulting in an increase of the pH [48]. We measured the secretion of ammonia in vitro at pH �5 by the two strains and found that 398 produces significantly higher amounts of ammonia than 19606 (Fig 7D) at earlier time points. Furthermore, we detected higher levels of intracellular ammonia in macrophages infected with 398 compared to non-infected cells or cells infected with 19606 ( Fig 7E). Employing additional strains, we observed that intracellular replication correlates with the ability of the strains to withstand acidic pH and to produce ammonia in vitro (S12 Fig). Together, our results show that

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Interference of phagosomal maturation by Acinetobacter baumannii tolerance to acidic pH and the subsequent production and secretion of ammonia to neutralize the intravacuolar pH are key for A. baumannii replication within the ACV.

Discussion
Here, we demonstrate that classic A. baumannii lab strains, like 19606, and recent clinical isolates, such as 398, behave differently, both in vivo and in vitro. While 398 was able to replicate in large ACVs within AMs during murine respiratory infection, 19606 was quickly cleared by these cells . Fig 8 summarizes our findings related to the intracellular behavior of both replicative and non-replicative strains in vitro. Initially, both strains follow the canonical endocytic pathway, as determined by the sequential colocalization of EEA1 and LAMP1 markers with phagosomes. 19606 phagosomes continue with their maturation and fuse with autophagosomes and lysosomes to ultimately be degraded by the macrophage. However, our data show that 398 ACVs avoid the autophagic and degradative pathways allowing the bacteria to survive and replicate inside macrophages. Moreover, 398's ability to quickly grow at acidic pH allows this strain to revert the initial luminal acidification of the vacuole, most likely via production

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Interference of phagosomal maturation by Acinetobacter baumannii of ammonia, a by-product of amino acid degradation. The process generates a non-degradative niche that allows replication of A. baumannii inside the ACV.
Several reports have established the critical role of AMs during A. baumannii respiratory infections [5,7,49,50]. Depletion experiments demonstrated that AMs are necessary to control tissue damage, bacterial sepsis and severity of the infection in an A. baumannii pneumonia models [7]. Recent reports showed that A. baumannii modern clinical isolates are able to survive and/or replicate intracellularly, in vitro, in macrophages [33,34] and lung epithelial cells [32]. To the best of our knowledge, this is the first demonstration of the presence of large vacuoles containing A. baumannii within AMs in a mammalian pneumonia model. A similar behavior has been described for other pathogens, such as M. tuberculosis, Staphylococcus aureus, and L. pneumophila, in which bacteria use alveolar macrophages as a reservoir to persist and replicate within the host [51][52][53][54]. Therefore, replication in AMs could be a key factor for the persistence of A. baumannii within the infected hosts.
We demonstrated that the intracellular compartment produced by replicative A. baumannii strains such as 398 was clearly different than the classical phagosome formed by non-replicative strains such as 19606. Avoiding fusion with autophagosomes is critical for some intracellular bacteria to escape from degradation. The interaction between A. baumannii and autophagy has been studied in different host cells, but the conclusions were disparate. In epithelial cells, non-replicative A. baumannii strains induce autophagy, leading in some cases to persistence [55,56], or degradation [44,57]. In THP-1 monocytes or MH-S macrophages, induction of autophagy after infection with A. baumannii strain 98-37-09 leads to bacterial killing by the host cell [57]. Moreover, LC3 colocalized with A. baumannii strains AB5075 and 98-37-09 internalized by epithelial cells [44,57]. However, our data is in agreement with recent works that show that new clinical isolates that replicate inside ACVs in epithelial cells or macrophages do not recruit LC3 or get degraded [32,33].
Phagosomal acidification, produced mainly by the accumulation of V-ATPases, is critical to eliminate some bacteria [20]. Several intracellular pathogens have developed strategies to avoid phagosomal acidification [58,59]. Here, we observed that initially the ACV of the replicative isolate 398 is acidic; however, as the infection progresses, the strain can actively increase the internal pH of the compartment. Moreover, we determined that A. baumannii replicative strains can better withstand acidic conditions and grow to a faster rate than non-replicative strains. This allows these strains to release higher levels of ammonia, leading to a quicker neutralization of the vacuolar pH (Figs 7 and S12). While most prominent Gram-negative intracellular pathogens use a type III or IV secretion system [60,61] to manipulate the host cell machinery, A. baumannii only has a Type I secretion systems (T1SS) and Type II secretion systems (T2SS) [62]. Multiple reports have established that the presence of ammonia in the phagosome inhibits the fusion of this compartment with lysosomes in macrophages [63][64][65]. Other pathogens such as Cryptococcus neoformans or Helicobacter pylori can increase the phagolysosomal pH expressing a urease to produce ammonia [66,67]. M. tuberculosis secretes the asparaginase AnsA and avoids phagosomal acidification by the production of ammonia, enabling intracellular replication [68]. We propose a model in which A. baumannii strains that resist the initial acidic stress of the phagosome are able to grow and release ammonia as a metabolic by-product, thereby neutralizing the microenvironment of the ACV. Future work will determine if A. baumannii employs T1SS or T2SS effectors to aid in preventing acidification of the ACV and intracellular replication.
An increasing number of studies, including this one, demonstrate differences in intracellular behavior within macrophages among A. baumannii isolates [32][33][34]69,70]. Based on this evidence, we propose to redefine A. baumannii as a facultative intracellular bacterium that can grow outside and within the host cell [71]. From the results obtained in this work, it is evident

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Interference of phagosomal maturation by Acinetobacter baumannii that A. baumannii can develop ACVs in AMs in vivo in a murine pneumonia model and it is not a phenomenon restricted to in vitro observations. We recently demonstrated that the clinical isolate UPAB1 establishes small intracellular reservoirs, known as ABIR (A. baumannii intracellular reservoirs) in bladder epithelial cells of mice that previously cleared a urinary tract infection. We observed that these ABIRs likely act as reservoirs that can be activated upon insertion of a medical device, such a catheter, leading to a resurgent infection in mice [72]. It is tempting to speculate that intracellular A. baumannii in AMs may also function as reservoirs in the host. We hypothesize that this intracellular lifestyle of A. baumannii enhances persistence inside the host. Within AMs, A. baumannii is protected from the immune system and from antibiotics that do not penetrate eukaryotic cells, like aminoglycosides and polymyxins.
Here, we have demonstrated that A. baumannii strain 398 and possible some other recent clinical isolates, are able to manipulate the host intracellular environment to form a replicative niche. Future work will focus on the identification of additional factors that A. baumannii employs to survive inside these host cells, which may lead to improved therapeutic interventions to treat modern A. baumannii clinical isolates.

Bacterial strains and culture conditions
The bacterial strains employed in this work are described in S1 Table. Bacteria were seeded on lysogeny broth (LB) agar plates and incubated 16 h at 37˚C. Thereafter, colonies were grown in LB broth under shaking conditions (200 rpm) for 16 h at 37˚C. When appropriate, antibiotics were added to the cultures at the following concentrations: 50 μg/ml zeocin, 30 μg/ml kanamycin, and 15 μg/ml chloramphenicol.

Growth assays
A. baumannii strains were cultured overnight in LB broth at 37˚C under shaking conditions. Bacterial cultures were centrifugated at 6500 rpm for 5 min and the pellets were washed with Phosphate-buffered saline (PBS, Sigma, D8662). A. baumannii suspensions were diluted to OD 600 = 0.01 in 150 μl of LB buffered at pH 5 (MES 100 mM), 6 (MES 100 mM), or 7 (HEPES 100 mM) in 96-well plates and incubated 16 h at 37˚C under shaking conditions. OD 600 values were measured at 30 min intervals using a BioTek microplate spectrophotometer. The experiment was performed in biological triplicate with three technical replicates per experiment for each strain and condition.

pH measurements in A. baumannii cultures
The absorbance of phenol red solutions at 560 nm is an indicator of pH, as previously described [73][74][75]. Phenol red is a pH indicator changing color from yellow below pH 6.8 to bright pink above pH 8.2. To analyze the pH changes in A. baumannii cultures during bacterial growth, indicated strains were maintained overnight in LB broth at 37˚C under shaking conditions. Bacterial cultures were washed and diluted as described for growth assays, with the following modification: non-buffered LB pH 5 with phenol red (15 μg/ml) was used. OD 600 and OD 560 values were measured every 30 min for 16 h using a BioTek microplate spectrophotometer. The OD 600 absorbance values were subtracted from the absorbance at 560 nm data to exclude the effect of bacterial density. Finally, the values obtained were used to estimate culture pH by employing a standard curve of OD 560 vs pH. The experiment was performed in triplicate with three technical replicates per each strain.

Murine Model of A. baumannii pneumonia
Colonies from A. baumannii strains 398, GFP-398 and GFP-19606 were subcultured from LB agar plates and grown in LB broth 16 h at 37˚C under shaking conditions. Overnight cultures obtained were diluted in sterile LB broth (1:100) and incubated for 3 h at 37˚C under shaking conditions. The bacterial inocula were prepared by centrifugation at 6500 rpm for 5 min, washed twice in PBS, and the pellet resuspended in PBS. Six-to eight-week-old female C57BL/ 6 mice obtained from Charles River Laboratories were intranasally inoculated with A. baumannii strains 398, GFP-398, or GFP-19606. Briefly, mice were anesthetized by inhalation of 4% isoflurane and were infected immediately with 50 μL of bacterial suspension (*1 × 10 8 Colony Forming Units [CFU]) that were gradually released with a micropipette into the nares. At 3 and 24 hours post-infection (hpi), bronchoalveolar lavage fluid (BALF) was collected. For BALF collection, mice were euthanized by CO 2 asphyxiation and then, the lungs and trachea were exposed by surgical dissection and polyethylene tubing (0.86x1.27mm) was inserted into the trachea. Cold PBS containing 1mM EDTA was flushed through the lungs in 1ml increments to collect a total volume of 10 ml. All pneumonia studies were performed in accordance with the guidelines of the Committee for Animal Studies at Washington University School of Medicine, and we have complied with all relevant ethical regulations.

Antibiotic protection assay
To analyze the intracellular replication of the different strains, J774A.1 cells were seeded 16 h before the experiment in 48-well plates (3x10 5 cells/well). A. baumannii colonies from LB agar plates were inoculated in LB broth and grown overnight at 37˚C under shaking conditions. Bacterial cultures were centrifugated 5 min at 6500 rpm and washed twice with PBS. Then, bacterial cultures were normalized to OD 600 � 1 and appropriate volumes was added to 500 μl of DMEM per well to achieve an MOI�10. Where indicated, bafilomycin A1 (15 nM; Sigma, B1793) was added to the growth media and maintained throughout the infection. Infected cells were centrifugated 10 min at 200 x g and incubated at 37˚C and 5% CO 2 . After 1 h, macrophages were washed three times with PBS and treated with DMEM supplemented with 10% FBS and colistin (50 μg/mL) to eliminate the extracellular bacteria. At the time points indicated, the cells were washed three times with PBS and lysed with 500 μl of Triton X-100 (0.05%) per well. Serial dilutions of bacterial suspensions obtained were plated on LB agar and incubated overnight at 37˚C, to determine number of colony forming units (CFUs).
To determine the number of total and intracellular bacteria present in BALF from A. baumannii infected mice, two 500 μl aliquots of lavage fluid were centrifuged at 6500 rpm for 10 min. The pellets were incubated for 1 h at 37˚C in PBS (total bacteria) or PBS with colistin (50 mg/mL) (intracellular bacteria). Then the cells were washed three times with PBS and lysed with 500 μl of Triton X-100 (0.05%) per aliquot. CFUs were determined by serial dilutions of the bacterial suspensions.

Ammonia production
Ammonia production by A. baumannii strains was measured using the Ammonia Assay Kit (AA0100, Sigma) according to the manufacturer's instructions. Briefly, the strains were

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Interference of phagosomal maturation by Acinetobacter baumannii cultured overnight in LB broth at 37˚C under shaking conditions. Then, bacterial cultures were centrifugated, and washed with PBS. A. baumannii suspensions were diluted to OD 600 = 0.01 in 150 μl of buffered LB (MES 100 mM, pH 5) in 96 well plates and incubated 6 h at 37˚C under shaking conditions. Ammonia levels were measured, at the indicated times. The experiment was performed in biological triplicate.
Intracellular ammonia production was measured using the same kit. Briefly, macrophages were infected with A. baumannii strains 19606 or 398 in duplicates. 24 hpi a set of wells was scraped in PBS to determine the number of macrophages. The remaining wells were treated with 200 μl of Triton X-100 (0.05%), centrifuged at 6500 rpm for 10 min and ammonia levels were measured in the supernatant.

Immunofluorescence staining
1.3x10 5 J774A.1 cells were plated onto glass coverslips in 24 well plates and incubated 16 to 18 h at 37˚C and 5% CO 2 . Inocula of the indicated A. baumannii strains were prepared by centrifugation of overnight cultures at 6500 rpm for 5 min, washing twice in PBS, and resuspension of the pellet in PBS. Bacterial suspensions were normalized to OD�1 and an appropriate volume was used to infect the cells (MOI�10). Afterward, the plates were centrifugated 10 min at 200 x g to enhance bacterial contact with the host cells and incubated for 1 h at 37˚C and 5% CO 2 . Cells were washed three times with PBS, and extracellular bacteria were killed by

Confocal microscopy
Infected cells were analyzed with a Zeiss LSM880 laser scanning confocal microscope (Carl Zeiss Inc.) equipped with 405nm diode, 488nm Argon, 543nm HeNe, and 633nm HeNe lasers. A Plan-Apochromat 63X (NA 1.4) DIC objective and ZEN black 2.1 SP3 software were used for image acquisition. Live images were acquired with a Zeiss spinning disk confocal microscope (Carl Zeiss Inc.) equipped with 488nm and 560nm lasers. A Plan-Apochromat 63X/1.3 Oil Ph 3 (UV) VIS-IR M27 objective and ZEN black 2.1 SP3 software were employed for image acquisition. Images were analyzed using ImageJ software (NIH, USA).
Live imaging 5x10 5 J774A.1 macrophages were plated in a 10 mm Glass Bottom Culture 35 mm petri dish (MATEK corporation, P35G-0-14-C) and incubated 12-16 h at 37˚C and 5% CO 2 . The next day, cells were infected with mCherry-398 at an MOI�10 as described above. 15 minutes prior to the indicated infection time points, cells were washed twice with PBS and incubated in DMEM with LysoSensor Green DND-189 (1μM; Molecular Probes, Invitrogen, L7535). The cells were then rinsed twice in PBS and were immediately analyzed by confocal microscopy. During image acquisition, the cells were maintained at 37˚C and 5% CO 2 in a temperaturecontrolled CO 2 chamber on the microscope.

Transmission electron microscopy
Cells from BALF were centrifuged at 300 x g for 5 minutes and incubated in 500μl Pharm Lyse Buffer (BD Biosciences 555899) for 3 minutes at room temperature to lyse red blood cells. The cells were centrifugated and resuspended in PBS. Total cells number and viability were measured using Trypan Blue solution (Sigma T8154) and counted with TC20 automated cell counter (BioRad). At least 1x10 6 cells were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM sodium cacodylate buffer pH 7.2 for 1 h at room temperature and subsequently incubated to 4˚C overnight. Samples were washed in sodium cacodylate buffer at room temperature and postfixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. The cells were then rinsed in distilled water, and bloc-stained for 1 h with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA). Subsequently, the samples were rinsed in distilled water several times, dehydrated in a graded series of ethanol, and finally embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope

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Interference of phagosomal maturation by Acinetobacter baumannii (JEOL USA Inc., Peabody, MA) equipped with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA). Images were processed using ImageJ software.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA). Datasets were analyzed by Mann-Whitney test, Welch's t-test, one way ANOVAtest or two-way ANOVA-test, as indicated.

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Interference of phagosomal maturation by Acinetobacter baumannii S1